MS

Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 283–295

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Chemical profiles and quality evaluation of Buddleja officinalis flowers by HPLC-DAD and HPLC-Q-TOF-MS/MS Guoyong Xie 1 , Qiuhong Xu 1 , Ran Li, Lu Shi, Yu Han, Yan Zhu, Gang Wu ∗ , Minjian Qin ∗ Department of Resources Science of Traditional Chinese Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 210009, China

a r t i c l e

i n f o

Article history: Received 24 April 2018 Received in revised form 22 July 2018 Accepted 17 October 2018 Available online 22 October 2018 Keywords: Buddleja officinalis Chemical constituents HPLC-Q-TOF-MS/MS HPLC fingerprint

a b s t r a c t The dried flowers and inflorescences of Buddleja officinalis Maxim are used as traditional medicines in China, and aqueous extracts of the flowers have also been used since ancient times as a yellow rice colorant at local festivals. In this study, HPLC-Q-TOF-MS/MS was used to determine the overall chemical composition of this medicine-food plant. A total of 54 compounds, including 23 flavonoids, 19 phenylethanoid glycosides, 7 alkaloids and 5 other compounds, were detected in the methanol extracts of the herb using this method. Among them, 35 compounds were found firstly in this herb. HPLC fingerprints were also developed, together with a method for the simultaneous quantification of 11 constituents that could be used for quality evaluation of B. officinalis. Fingerprint analysis, using 28 characteristic fingerprint peaks, was used to assess the similarities among 12 samples collected from different geographic areas and showed that the similarity was >0.900. Simultaneous quantification of 11 markers in B. officinalis was then performed to determine consistency of quality. Additionally, the total phenolic content and antioxidant capacity of extracts of the 12 samples of B. officinalis flowers were measured using spectroscopic methods. B. officinalis was found to have good antioxidant capacity and to be a potential natural antioxidant. The highest antioxidant capacity was found in the samples from Guizhou, Sichuan and Guangxi Province. Our results provide valuable information for further understanding and exploiting the herb. © 2018 Elsevier B.V. All rights reserved.

1. Introduction The genus Buddleja (Loganiaceae), which comprises about 100 species, is widely distributed in the tropical and subtropical regions of America, Africa and Asia, with 20 species and five hybrids found in China [1]. Buddleja officinalis Maxim., known as Mi Meng Hua in Chinese, is a shrub that grows in Myanmar, Vietnam and in the southwest and central regions of China. The plant was first recorded 1000 years ago in Kai Bao Ben Cao, one of the ancient works on traditional Chinese medicines, and is now listed in the Chinese Pharmacopoeia (version 2015) [2]. The dried flower buds and inflorescences of B. officinalis are used as a traditional herbal medicine to treat eye diseases, strokes, vascular diseases, diabetes and neurological disorders [3,4]. The plant is also cultivated as a natural food dye and aqueous extracts of the flowers have been used since

∗ Corresponding authors at: Department of Resources Science of Traditional Chinese Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, No. 24 Tongjiaxiang, Gulou District, Nanjing, 210009, China. E-mail addresses: [email protected] (G. Wu), [email protected] (M. Qin). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jpba.2018.10.030 0731-7085/© 2018 Elsevier B.V. All rights reserved.

ancient times as a yellow rice colorant in local festivals in southwest China [5]. Phytochemical studies have demonstrated that the flowers of B. officinalis contain mainly flavonoids, phenylethanoid glycosides, iridoids and saponins [6–8], although a chemical profile reflecting the total constituents of the herb is not yet available. High performance liquid chromatography, coupled with diodearray detection (DAD) and high resolution time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS), can simultaneously separate and identify chemical constituents in medicinal herbs [9–11] and should be an effective tool for establishing chemical profiles of extracts of B. officinalis flowers (BOEs). It is said in Xin Xiu Ben Cao, “Supposing that the herb is away from its native land, then its effects will be different even if the features are identical”, and modern research has shown that the quality of traditional Chinese medicine (TCM) is mainly affected by the ecological environment, including soil, air temperature, rainfall, light and ecological communities. Beyond that, different harvest and storage conditions are important influencing factors [12]. Currently, the fingerprint technique is acknowledged by the U.S. Food and Drug Administration (FDA), the World Health Organization (WHO) and the State Food and Drug Administration of China (SFDA) for the quality control of TCM [13]. HPLC, thin layer

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Table 1 Source information of 12 batches samples and their calculated similarity values. No. a

Collection site (Province)

Collect date

Similarity

No.a

Collection site (Province)

Collect date

Similarity

S1 S2 S3 S4 S5 S6

Anhui Guizhou Sichuan Tibet Yunnan Shaanxi

2016.01 2016.01 2016.10 2016.03 2016.03 2016.03

0.955 0.998 0.999 0.969 0.999 0.995

S7 S8 S9 S10 S11 S12

Shanxi Jiangsu Hunan Inner Mongolia Gansu Guangxi

2016.03 2016.03 2016.03 2016.03 2016.03 2016.03

0.998 0.996 0.999 0.998 1 0.993

a

All samples were dried in the sun.

chromatography (TLC) and gas chromatography (GC) are recognized as conventional analytical methods and, of these, HPLC has become the preferred method for TCM fingerprinting because of its rapidity, high separation efficiency, high selectivity, high sensitivity and wide applicability [14]. In the present study, we aimed to establish a chromatographic fingerprint for B. officinalis using HPLC-DAD. Using the method for simultaneous determination of 11 constituents established in our earlier study [15] as a foundation, we also analyzed the constituents of 12 samples of B. officinalis from different regions of China. Free radicals formed during metabolism are closely linked with aging and with diseases such as cancer, atherosclerosis and eye disease [16]. Since antioxidants are associated with reduced oxidative damage, measurement of the antioxidant capacity of medicines and food products is important for the health-related and nutraceutical industries [17]. B. officinalis flowers, which are used as a traditional herbal medicine and as a rice colorant for festivals, possess many biological and pharmaceutical activities, including anti-inflammatory, antioxidant, hypoglycemic, antiviral and antibacterial effects [18]. Previous studies have shown that B. officinalis flowers have pronounced antioxidant activity and show good potential as a natural antioxidant resource [18–22]. In the present study, the antioxidant capacities of 12 samples were measured to evaluate the antioxidant capacity of B. officinalis. To determine the overall chemical composition and identify antioxidant components of B. officinalis flowers, an HPLC-QTOF-MS/MS method was developed for qualitative analysis. The chromatographic fingerprint of B. officinalis was established and the main chemical components were quantified for samples of B. officinalis from 12 different producing areas. Anti-oxidative assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion • (O2 − ) scavenging activity (SOSA), Fe3+ -reducing ability (FRA) and Fe2+ -chelating assays were performed to evaluate the antioxidant capacity of B. officinalis. 2. Materials and methods 2.1. Materials and chemicals Flower buds and inflorescences of B. officinalis were collected from 12 habitats in China. Sample information is provided in Table 1. Sample S3 was selected for chemical profiling. The samples were crushed to a powder in an electric grinder and then passed through a 60 mesh sieve. Reference compounds, including luteolin 7-O-glucoside, luteolin 7-O-rutinoside, neobudofficide, linarin, apigenin, acteoside, crocin III and N1 ,N5 , N10 -(E)-tri-p-coumaroylspermidine, were obtained from B. officinalis flowers by the authors. Apigenin 7-O-glucuronide, acacetin, echinacoside and isoacteoside were purchased from Baoji Herbest Bio-Tech Co., Ltd. (Baoji, China). The purity of all standards was >96%, as determined by HPLC. HPLC grade methanol and acetonitrile were purchased from Hanbon Sci. & Tech. Co., Ltd. (Jiangsu, China) and Merck (Darmstadt, Germany), respectively. Purified water was obtained from Wahaha Group Co., Ltd. (Hangzhou, China). Gallic acid, 1,1-diphenyl-2-picrylhydrazyl

(DPPH) and nitrotetrazolium blue chloride (NBT) were purchased from Sigma-Aldrich Co. (Shanghai, China). Folin-Ciocalteu’s reagent, l-methionine and riboflavin were obtained from Solarbio Sciences & Technology Co., Ltd. (Beijing, China). Trichloroacetic acid (TCA) and potassium ferricyanide (K3 Fe(CN)6 ) were purchased from Shanghai Macklin Reagent Company (Shanghai, China). Ferrozine was obtained from Aladdin Industrial Corporation (Shanghai, China). Sodium carbonate (Na2 CO3 ), Iron (III) chloride hexahydrate (FeCl3 ·6H2 O), Ferrous chloride (FeCl2 ), Disodium hydrogen phosphate dodecahydrate (Na2 HPO4 ·12H2 O) and Sodium dihydrogen phosphate dihydrate (NaH2 PO4 ·2H2 O) were purchased from China Pharmaceutical Group Chemical Reagent Co., Ltd. (Beijing, China). 2.2. Preparation of sample solutions and standard solutions The extraction condition was optimized in our previous studies [15], the dried powder (0.5 g) was sonicated twice for 33 min with 76% methanol (17 mL). The combined extracts were centrifuged at 1500 r/min for 10 min. The supernatant was then transferred to a 50 mL volumetric flask and the volume made up to 50 mL using the same solvent. Standard stock solutions of the 11 reference compounds were prepared by dissolving accurately weighed amounts in 60% aqueous acetonitrile. A mixed standard stock solution was then prepared using the individual standard stock solutions. All solutions were stored in a refrigerator at 4 ◦ C and filtered through 0.22 ␮m membrane filter before analysis. 2.3. Chromatographic conditions HPLC fingerprinting and composition analysis were conducted using a method established previously [15]. An Agilent Series 1260 LC instrument (Agilent Technologies, Santa Clara, CA, USA), equipped with autosampler, column temperature controller, DAD, quaternary pump and online degasser, was used for the analyses. A Megres C18 column (4.6 mm × 250 mm, 5 ␮m, Hanbon Sci. & Tech. Co., Ltd.), thermostated at 25 ◦ C, was used for chromatographic separations. The mobile phase was 0.1% formic acid-water (A) and acetonitrile (B). The gradient elution program was: 0–5 min, 8–10% B; 5–10 min, 10–18% B; 10–20 min, 18–18.5% B; 20–34 min, 18.5–30% B; 34–38 min, 30% B; 38–41 min, 30–37% B; 41–50 min, 37–95% B; 50–55 min 95% B. The flow rate was 1.0 mL/min and the detection wavelength was 330 nm. The injection volume was 10 ␮L and each run was followed by an equilibration period of 5 min. An Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA), equipped with autosampler, column temperature controller, DAD, quaternary pump and online degasser, was used for liquid chromatography separations. The chromatographic conditions were the same as those used for HPLC fingerprinting and composition analysis. In order to identify the constituents, the HPLC system was coupled to an accurate-mass Q/TOF-MS/MS instrument (Agilent Technologies, Santa Clara, CA, USA), operating in positive and negative electrospray ionization (ESI) mode. The parameters for MS detection were as follows: scan range from m/z 100–2000 Da, nitrogen flow rate 8 L/min, drying gas temperature 325 ◦ C, nebu-

G. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 283–295

lizer pressure 50 psi and capillary voltage 3500 V. Operation, data capture and analysis were managed using Masshunter Qualitative Analysis Software B.06.00 and ChemStation software. 2.4. Method validation for HPLC fingerprinting Method validation for HPLC fingerprinting includes precision, stability and reproducibility tests. The precision was evaluated by analyzing the same sample solution (sample 3) five times continuously; The stability was determined by analyzing one sample solution (sample 3) after storage at 4 ◦ C for 0, 2, 4, 6, 8, 12, 24 and 48 h, and the reproducibility was assessed by injection of five working solutions (sample 3).

Total phenolic content (TPC) was determined using the FolinCiocalteau reaction [23]. Extract solution (200 ␮L, 5 mg/mL) was blended with Folin-Ciocalteau reagent (200 ␮L) and, after 5 min, 3% Na2 CO3 solution (7 mL) was added. The combined reaction solution was stored at room temperature for 1 h, and then determined at 765 nm using an Epoch microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). The standard curve (y = 0.0013 x + 0.0761, R2 = 0.999) was established using gallic acid as reference. Results are expressed as gallic acid equivalents (GAE) (mg)/dry mass of plant material (g). 2.6. Radical scavenging activity 2.6.1. DPPH radical scavenging activity DPPH radical scavenging capacity was measured using a previously reported method [24]. A working solution of DPPH (0.05 Mm/L) was prepared by dilution with methanol. Sample solutions (200 ␮L) at different concentration (1.5, 1, 0.75, 0.5, 0.25, 0.125 mg/mL) were then mixed with DPPH solution (3 mL) and the reaction mixtures were allowed to stand for 30 min in the dark. By measuring absorbance at 517 nm, DPPH radical scavenging activity was calculated using the following formula:



Scavenging rate (%) = 1 − (Atest − Ablank ) /Acontrol × 100% where Atest is the absorbance of the solution of sample and DPPH after reaction, Ablank is the absorbance of the sample and 76% methanol (3 mL) and Acontrol is the absorbance of 76% methanol (200 ␮L) and DPPH. IC50 values were calculated for evaluation of the DPPH radical scavenging activity of the samples, Vc (vitamin C) used as the positive reference. •

2.6.2. Superoxide anion (O2 − ) scavenging activity SOSA was analyzed as previously described [25]. The reaction was carried out at 25 ◦ C and the final assay mixture contained lmethionine solution (130 mM/L, 0.3 mL), NBT solution (750 ␮M/L, 0.3 mL), EDTA-Na2 solution (20 ␮M/L, 0.3 mL) and riboflavin solution (20 ␮M/L, 0.3 mL). The assay mixture was added to 50 mM/L phosphate buffer (pH 7.0, 1.5 mL) and mixed with different concentrations (750, 500, 375, 250, 125, 62.5 ␮g/mL) of test sample (0.2 mL). The test sample was replaced by the extraction solvent (76% methanol) in the control group and blank group. After exposure of the test and control solutions to fluorescent light for 30 min, absorption was measured at 560 nm. SOSA was calculated using the following formula:



the blank solution (lucifuge). Results are expressed as IC50 values (␮g/mL), Vc was used as the positive reference. 2.6.3. Fe3+ -reducing ability FRA was measured as previously described [26]. Different concentrations (2.5, 2, 1.5, 1, 0.5, 0.25 mg/mL) of sample solutions (500 ␮L) were mixed with phosphate buffer (0.5 mL, 0.2 M/L, pH 6.6) and K3 Fe(CN)6 (0.5 mL, 1%). The mixtures were heated in a water bath at 50◦ for 20 min, TCA (0.5 mL, 10%) was then added and the mixtures were centrifuged at 4000 r/min for 10 min. An aliquot (0.5 mL) of the upper layer was mixed with distilled water (0.5 mL) and 0.1% FeCl3 (0.1 mL), and the absorbance was measured at 700 nm. FRA was calculated using the following formula: Fe3+ reducingability(A) =Atest −Ablank

2.5. Determination of total phenolic content



285



Scavenging rate (%) = 1 − (Atest − Ablank ) /Acontrol × 100% where Atest is the absorbance of the test solution, Acontrol is the absorbance of the control solution and Ablank is the absorbance of

where Atest is the absorbance of the test solution, Ablank is the absorbance of the blank solution (76% methanol instead of sample). EC50 values were calculated for evaluation of FRA of the samples, Vc used as the positive reference. 2.6.4. Fe2+ -chelating assay Fe2+ -chelating capacity was measured as previously described [27]. Different concentrations (10, 8, 6, 4, 2, 1 mg/mL) of sample solution (400 ␮L) were added to a mixture of aqueous ferrous chloride solution (2 mM/L, 50 ␮L), ferrozine solution (5 mM/L, 100 ␮L) and methanol (2 mL). After shaking vigorously, the reaction solutions were allowed to stand at 25◦ for 10 min. Absorbance was measured at 562 nm and Fe2+ -chelating capacity was calculated using the following formula:





Inhibition rate (%) = 1 − (Atest − Ablank ) /Acontrol × 100% where Atest is the absorbance of the test solution, Ablank is the absorbance with EDTA-Na2 , Acontrol is the absorbance without sample. Results are presented as IC50 values (mg/mL), EDTA-Na2 was used as the positive reference. 2.7. Data analysis The HPLC-DAD data from 12 samples of B. officinalis were analyzed using the professional software, Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004 A), which is recommended by the SFDA. The mean chromatogram was calculated and generated as a representative standard fingerprint chromatogram and the correlation coefficients of the samples were then calculated by comparison with the mean chromatogram. All experiments were analyzed in triplicate. Data treatment was carried out using Microsoft Excel software and SPSS software 22.0. 3. Results and discussion 3.1. Identification of chemical constituents in methanol extract of B. officinalis flowers HPLC-ESI-Q/TOF-MS/MS analysis of the methanolic BOEs was carried out in both positive and negative ionization modes. The HPLC chromatograms, together with the total ion currents (TIC) of the methanolic BOEs, are shown in Fig. 1 and the UV and MS data are listed in Table 2. Fifty-four compounds were identified or tentatively identified, including 23 flavonoids, 19 phenylethanoid glycosides, 7 alkaloids and 5 other compounds. The chemical structures of the compounds are shown in Fig. 2. The peaks for compounds F4, F5, F11, F14, F16, F22, F23, P10, P16, A6 and C5 were definitively identified by comparison of UV and MS data with those of authentic standards. The chemical structures

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Fig. 1. The total ion chromatograms of authentic standards and methanol extract of Buddleja officinalis flowers. (A) UV chromatogram of the 11 authentic standards at 330 nm; (B) UV chromatogram of methanol extract at 330 nm; B(n) and B(p), TIC chromatograms of methanol extract in negative and positive ion mode respectively; The peak numbers are the same as those of the compounds in Table 2 and Fig. 2.

of other compounds were deduced mainly on the basis of the UV and MS data and confirmed by comparison with values reported in the literature. Analysis of the BOEs revealed that flavonoids and phenylethanoid glycosides were the major constituents in B. officinalis flowers. 3.1.1. Flavonoid compounds Twenty-three flavonoids, including 3 free aglycones and 20 Oglycosides, were identified in the BOEs. Retro-Diels-Alder cleavage of the C-ring, combined with various neutral loss pathways, are typically used for the identification of flavonoid aglycones [28]. For the identification of flavonoid glycosides, ion fragmentations are characterized by successive elimination of sugar moieties and aglycone fragments [29]. In this study, the major flavonoid aglycones in the BOEs were identified as luteolin (F20), apigenin (F22) and acacetin (F23) by comparison with authentic standards and literature data [30,31]. Of the 20 flavonoid O-glycosides, all except F3, F9 and F12 were identified as derivatives of these three aglycones. Compounds F1, F2, F7, F8, F10, F11, F13 and F15 were identified as apigenin (F22) derivatives. F11 was unambiguously confirmed to be apigenin 7-O-glucuronide by comparison with the authentic standard compound and with literature data [32]. F1 and F2 showed identical molecular ions and similar fragmentation patterns in positive ionization mode. Their MS2 spectra exhibited ions at m/z 447 [M+H-176]+ and 271 [M+H-176-176]+ , which originated from successive losses of 176 Da fragments, indicating the presence of two glucuronic acid moieties. However, F1 and F2 showed different fragmentation behaviors in negative ionization mode. The spectrum of F2 showed a fragment ion at m/z 269 [M−H-352]¯, whereas this fragment was absent in the spectrum of F1. Taking into account the MSn and UV spectra, together with previous reports, F1 and F2 were tentatively deduced to be apigenin 7,4 di-O-glucuronide and clerodendrin, respectively [33,34]. Compared with F2, F8 exhibited fragment ions at m/z 447 [M+H-146]+ and 271 [M+H-146-176]+ in positive ionization mode, indicating that a glucuronic acid moiety was replaced by a rhamanopyranosyl group. Considering previously reported structural information [35], F8 was tentatively deduced to be apigenin 7-O-␣-l-rhamnopyranosyl(1→2)-ˇ-d-glucuronide. F7 gave a molecular ion at m/z 579 [M+H]+ , with product ions at m/z 433 [M+H-146]+ and 271 [M+H-146-162]+ , corresponding to the sequential loss of rhamanopyranosyl and glucose moieties. F7 was tentatively deduced to be isorhoifolin by comparing the MS2 and UV data with literature values [36]. F10 presented a precursor ion [M+H]+ at m/z 433, which indicated a molecular formula C21 H20 O10 . The fragment ion at m/z 271 [M+H−162]+ , which represented the aglycone fragment of apigenin, was formed by loss of a glucose moiety from the precursor ion. By comparison with

literature data, F10 was tentatively deduced to be apigenin 7-Oglucoside [31]. F15 showed similar fragmentation to F10 in positive ionization mode. The fragment ion at m/z 433 [M+H-86]+ indicated loss of a malonyl residue. The direct loss of 247 Da indicated that the malonyl residue was attached on the glucose moiety. Therefore, F15 was tentatively identified as apigenin 7-(6”-malonylglucoside) [37]. F13 was an isomer of F11 and showed an identical molecular ion and fragmentation pattern to that of F11. MS2 fragmentation of both compounds presented the same ions at m/z 271 [M+H−176]+ and 269 [M−H–176]− in positive and negative ionization modes, respectively, attributed to loss of a glucuronic acid residue from the precursor ion and the deprotonated molecular ion. F13 was, therefore, identified as an apigenin monoglucuronide. Additionally, based on published data on the elution order of apigenin monoglucuronides [30,38], F13 was tentatively identified as apigenin 4 -O-glucuronide. F4, F5 and F6 were identified as derivatives of luteolin (F20). F20 gave molecular ions at m/z 287 [M+H]+ and 285 [M−H]− , respectively, in positive and negative ionization modes. In the MS2 spectrum, the molecular ions yielded specific fragments at m/z 151, 133 and 153, 135, respectively, which are characteristic of Retro-Diels-Alder fragmentations for luteolin. F20 was, therefore, tentatively identified as luteolin by comparison with literature data [39]. F4 and F5 were unambiguously identified as luteolin 7O-rutinoside and luteolin 7-O-glucoside by comparison with the authentic standards and their corresponding UV and MS spectra [31,40]. In positive ionization mode, F6 showed a precursor ion at m/z 463 [M+H]+ , which gave similar fragmentation and the same MS2 fragments ions, at m/z 287, 269 and 241, as F5. The fragment at m/z 287 corresponded to the [M+H−176]+ ion, indicating the presence of a glucuronic acid moiety. By comparing the characteristic fragment ions with those of previous reports [31,40], F6 was tentatively identified as luteolin 7-O-glucuronide. F14, F16–F19 and F21 were identified as derivatives of acacetin (F23). F14 and F16 were unambiguously identified as neobudofficide and linarin by comparison with reference standards and previous reports [41,42]. F17 exhibited precursor and deprotonated molecular ions at m/z 607 [M+H]+ and 605 [M−H]− , respectively. In positive ionization mode, the product ions at m/z 461 [M+H-146]+ and 285 [M+H-146-176]+ corresponded to sequential loss of the rhamanopyranosyl and glucuronic acid moieties. The MS2 spectrum showed fragments at m/z 283 [M−H-146-176]− and 268 [M−H-146-176-15]− in negative ionization mode. F17 was tentatively identified as acacetin 7-O-␣-l-rhamnopyranosyl(1→2)-ˇ-d-glucuronide or its isomer, which had not been previously described. F18 was an isomer of F16. In positive ionization mode, its main fragment ion was at m/z 285, corresponding

Table 2 Characterization of chemical constituents of B. officinalis by HPLC–DAD-Q-TOF-MS/MS. No

tR (time)

UV(nm)

Quasimolecular(n)[M−H] =[M + Cl/COOH]¯ (Error, ppm)

Quasi-molecular(p) [M+ H/Na/NH4 ]+ (Error, ppm)

Molecular formula

Molecular formula generator

MS/MS fragments (n)

MS/MS fragments (p)

Proposed compound

F1b F2b F3b

18.014 18.665 21.643

268, 322 268, 318 nd

nd 621.1106 (−1.28) 463.0892 (−1.18)

623.1253 (−0.74) 623.1249 (−1.32) 465.1015 (2.56)

C27 H26 O17 C27 H26 O17 C21 H20 O12

622.1170 622.1170 464.0955

nd 269 301, 229, 149

Apigenin 7,4’-di-O-glucuronide Clerodendrin Quercetin 7-O-glucoside

F4a F5a F6b

26.429 28.513 29.086

254, 266 (sh), 348 254, 266 (sh), 348 242, 266 (sh), 342

593.1518 (−1.32) 447.0927 (125) 461.0722 (1.23)

595.1668 (−1.28) 449.1089 (−2.26) 463.0865 (1.77)

C27 H30 O15 C21 H20 O11 C21 H18 O12

594.1585 448.1006 462.0798

F7 F8b

31.274 32.238

234, 266 334 238, 328

613.1327 (1.04) 591.1342 (2.34)

579.1718 (−2.00) 593.1497 (0.98)

C27 H30 O14 C27 H28 O15

578.1636 592.1428

285, 259 285, 255 357, 285, 243, 133 431, 269 269

447, 271 447, 271 303, 285, 273, 243, 177 449, 287, 235 287, 269, 257, 241 287, 269, 241 433, 271, 129 575, 447, 271

F9b F10 F11a F12b F13b F14a ,b F15b F16a F17b

32.759 33.332 34.140 34.244 34.687 37.318 38.204 40.054 40.887

240, 328 238, 268, 328 236 (sh), 266, 336 nd 234, 328 232, 268, 332 240,322 230, 268, 332 240, 268, 330

607.1675 (-0.84) 431.0977 (2.04) 445.0785 (-1.54) 533.0943 (-0.28) 445.0771 (1.57) 773.2056 (1.36) nd 627.1481 (-0.03) 605.1533 (-3.01)

609.1883 (−2.26) 433.1137 (−1.97) 447.0934 (−2.64) 535.1093 (−2.31) 447.0926 (−1.09) 739.2459 (−1.63) 519.1128 (−1.31) 593.1867 (−0.93) 607.1662 (−0.33)

C28 H32 O15 C21 H20 O10 C21 H18 O11 C24 H22 O14 C21 H18 O11 C34 H42 O18 C24 H22 O13 C28 H32 O14 C28 H30 O15

608.1741 432.1056 446.0849 534.1010 446.0849 738.2371 518.1060 592.1792 606.1585

299, 284 269, 268 269 489, 285 269 283 nd 283 283, 268

463, 301, 286, 258 271, 243 271 287 271 593, 447, 395, 285 433, 271 447, 133 461, 395, 285

F18b

41.044

240, 268, 332

nd

593.1868 (−0.57)

C28 H32 O14

592.1792

nd

447, 285, 133

F19b F20

43.962 44.066

nd 242, 268, 338

481.0892 (2.51) 285.0396 (3.16)

447.1296 (−1.58) 287.0553 (−1.04)

C22 H22 O10 C15 H10 O6

446.1213 286.0477

F21b

44.665

240, 320

459.0932 (0.96)

461.1087 (−1.63)

C22 H20 O11

460.1006

283, 268 257, 217, 175, 151, 133 283, 268

F22a

47.114

240, 265, 336

269.0454 (0.68)

271.0590 (4.45)

C15 H10 O5

270.0528

285, 270, 242 269, 241, 179, 153, 135 285, 270, 257, 242, 133 243, 229, 153, 119

Isorhoifolin Apigenin 7-O-␣-L-rhamnopyranosyl(1→2)-ˇ-d-glucuronide Diosmetin 7-O-rutinoside Apigenin 7-O-glucoside Apigenin 7-O-glucuronide Kaempferol 3-O-(6”-malonylglucoside) Apigenin 4’-O-glucuronide Neobudofficide Apigenin 7-(6”-malonylglucoside) Linarin Acacetin7-O-␣-l-rhamnopyranosyl(1→2)-ˇ-d-glucuronide or its isomer 5-[[6-O-(6-deoxy-˛-l-mannopyranosyl)-ˇ-dgalactopyranosyl]oxy]-7-hydroxy-2-(4methoxyphenyl)-4H-1-Benzopyran-4-one Acacetin 7-O-glucoside Luteolin

a

50.137

268, 328

283.0614 (−0.53)

285.0757 (−0.06)

C16 H12 O5

284.0685

P1 P2 P3b

11.892 12.390 12.650

222, 280 223,328 nd

335.0905 (−0.43) 487.1434 (4.55) 487.1434 (4.89)

323.1099 (1.72) 511.1433 (−4.58) 511.1411 (0.85)

C14 H20 O7 C21 H28 O13 C21 H28 O13

300.1209 488.1530 488.1530

P4b P5 P6b

12.934 13.689 14.109

232,325 224, 264, 326 nd

505.1570 (-0.29) 417.1400 (1.44) 341.0872 (0.34)

507.1723 (−2.31) 395.1313 (1.23) nd

C21 H30 O14 C17 H24 O9 C15 H18 O9

506.1636 372.1420 342.0951

P7b P8b

14.549 15.542

236, 290 (sh), 326 240, 326

353.0875 (1.03) 555.1476 (2.56)

355.1020 (0.62) 521.1880 (−3.33)

C16 H18 O9 C22 H32 O14

354.0951 520.1792

P9b

16.268

238, 328

481.1475 (1.77)

469.1683 (−0.30)

C20 H30 O11

446.1788

P10a P11b

17.128 17.809

220, 328 234, 329

785.2514 (0.07) 639.1941 (-0.82)

804.2910 (1.47) 663.1910 (−1.47)

C35 H46 O20 C29 H36 O16

786.2582 640.2003

341, 179, 161 nd 179, 161, 135, 133 191, 161 519, 397, 355, 295, 235, 193, 175 445, 263, 221, 179, 161, 131 623, 461, 459 610, 487, 477, 461, 285, 179, 161

270, 257, 242, 177, 153, 133 163, 121 365, 163, 151 365, 163, 151 163, 145 233, 232, 218, 185 nd

Acacetin 7-O-glucuronide Apigenin Acacetin Salidroside Cistanoside F 4-O-(3,4-Dihydroxy-Z-cinnamoyl)-3-OLrhamnopyranosyl-d-glucose Hebitol II Syringin ˇ-4-caff ;eoylglucose

163, 145, 135, 117 321, 195, 177, 145, 117

3-Caffeoylquinic acid Globularitol

307, 163

2-phenylethyl 6-O-ˇ-d-glucopyranosyl-ˇ-d-Glucopyranoside Echinacoside Isocampneoside II

679, 471, 325, 163 325, 309, 181, 163

G. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 283–295

F23

241, 227, 151, 117 268, 240, 211, 151 179, 137, 119 179, 161, 135 179, 161, 135

Luteolin 7-O-rutinoside Luteolin 7-O-glucoside Luteolin 7-O-glucuronide

287

288

Table 2 (Continued) tR (time)

UV(nm)

Quasimolecular(n)[M−H] =[M + Cl/COOH]¯ (Error, ppm)

Quasi-molecular(p) [M+ H/Na/NH4 ]+ (Error, ppm)

Molecular formula

Molecular formula generator

MS/MS fragments (n)

MS/MS fragments (p)

Proposed compound

P12

18.330

234, 328

639.1930 (0.64)

663.1916 (−3.02)

C29 H36 O16

640.2003

610, 487, 477, 461, 285, 179, 161

325, 309, 181, 163

Campneoside II

P13 P14b

19.264 23.072

nd 244, 326

451.1376 (2.80) 755.2424 (-2.63)

439.1587 (−2.60) 774.2825 (−0.3)

C19 H28 O10 C34 H44 O19

416.1682 756.2477

307 325, 163

Phenethyl-ˇ-primeveroside Hebeoside

P15b

26.953

234, 330

623.1982 (0.27)

642.2397 (−0.14)

C29 H36 O15

624.2054

463, 325, 287, 163

Forsythoside A

P16a

27.497

220, 330

623.1969 (2.45)

642.2398 (−0.19)

C29 H36 O15

624.2054

325, 163

Acteoside

P17b P18a

28.200 30.441

235, 328 242, 326

623.1986 (-0.25) 623.1990 (-1.15)

647.1944 (0.92) 647.1944 (0.92)

C29 H36 O15 C29 H36 O15

624.2054 624.2054

501, 325, 163 501, 325, 163

Cis-acteoside Isoacteoside

P19b

36.136

220, 330

961.2982 (-0.69)

980.3406 (−0.78)

C45 H54 O23

962.3056

501, 339, 325, 181, 177, 163

Globusintenoside or Globusintenoside isomer

A1b

26.715

244, 298

nd

438.2392 (−0.43)

C25 H31 N3 O4

437.2315

623, 593, 461, 447, 179, 161, 133, 113 461, 315, 179, 161, 135 477, 461, 315, 179, 161 461, 179, 161 487, 461, 179, 161 799, 785, 767, 623, 487, 179, 161 nd

A2b

43.962

246, 296

598.2556 (0.73)

600.2711 (−0.64)

C34 H37 N3 O7

599.2632

nd

N1 , N5 -di-[(E)-p-coumaroyl]- spermidine or its isomers N1 -(E)-caffeoyl-N5 , N10 -di-p- (E)-coumaroyl spermidine

A3b

45.212

nd

nd

584.2758 (−0.50)

C34 H37 N3 O6

583.2682

nd

A4b

45.577

246, 316

nd

584.2741 (0.53)

C34 H37 N3 O6

583.2682

nd

A5b

45.733

252, 320

618.2368 (1.06)

584.2761 (−1.07)

C34 H37 N3 O6

583.2682

nd

A6a,b

46.098

234, 294, 306(sh)

618.2389 (−1.47)

584.2756 (0.07)

C34 H37 N3 O6

583.2682

582, 462, 342, 316, 145

A7b

46.332

246, 298, 310

nd

614.2855 (0.35)

C35 H39 N3 O7

613.2788

nd

C1 C2b C3b

9.964 15.669 34.921

220, 280 nd 242, 442, 464(sh)

375.1284 (2.32) 387.1664 (-0.44) 975.3712 (0.12)

394.1712 (−0.47) 411.1636 (−2.76) 999.3659 (1.54)

C16 H24 O10 C18 H28 O9 C44 H64 O24

376.1369 388.1733 976.3788

195, 183, 151 207 nd

C4b

38.282

240, 442, 464 (sh)

859.2948 (1.79)

837.3180 (−3.3)

C38 H54 O19

814.3259

nd

C5a

46.489

258, 434, 458 (sh)

687.2425 (0.65)

675.2625 (0.13)

C32 H44 O14

652.2731

nd

421, 292, 275, 247, 218, 204, 147 454, 436, 421, 308, 292, 275, 220, 204, 163, 147 438, 420, 318, 292, 275, 218, 204, 172, 147 438, 420, 318, 292, 275, 218, 204, 172, 147 438, 420, 318, 292, 275, 218, 204, 172, 147 438, 420, 318, 292, 275, 218, 204, 172, 147 468, 450, 438, 420, 322, 305, 292, 275, 234, 218, 204, 177, 147 197, 179, 165, 123 249 907, 675, 583, 365, 351, 347 745, 675, 583, 513, 421, 365, 351, 347, 259 583, 365, 351, 347, 307, 259

sh, shoulder peak; nd, not detected. a Authentic standards. b Detected in Buddleja officinalis for the first time.

N1 , N5 , N10 -(Z)-tri-p- coumaroylspermidine

N1 (Z)-N5 -(Z)-N10 -(E)-tri-p-coumaroylspermidine

N1 (E)-N5 -(Z)-N10 -(E)-tri-p-coumaroylspermidine

N1 , N5 , N10 -(E)-tri-p- coumaroylspermidine

Keayanidine A

6-methoxylcatalpol Tuberonic acid glucose Crocin I Crocin II

Crocin III

G. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 283–295

No

G. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 283–295

289

Fig. 2. Chemical structures of compounds identified in the extracts of Buddleja Officinalis Maxim. F1-F23, flavonoids; P1-P19, phenylethanoid glycosides; A1-A7, alkaloids; C1-C5, other compounds.

to [M+H-146-162]+ , formed by loss of the rhamanopyranosylglucosyl moiety. The fragment ion at m/z 133, which represented the aglycone fragment, was identical to that of acacetin [42]. F18 was, therefore, tentatively identified as 5-[[6-O-(6-deoxy-˛l- mannopyranosyl)-ˇ-d-galactopyranosyl]oxy]-7-hydroxy-2-(4methoxypheny-l)-4H-1-benzopyran-4-one [43]. The MS spectra of F19 and F21 showed [M+H]+ ions at m/z 447 and 461, respectively, and showed the same MS/MS fragments at m/z 285, 270 and 242. The fragment at m/z 285 corresponded to the [M+H−162]+ ion for F19 and the [M+H−176]+ ion for F21, indicating that F19 possessed an O-glycoside moiety, while F21 possessed a glucuronic acid moiety. By comparison with literature data [40], F19 and F21 were tentatively identified as acacetin 7-O-glucoside and acacetin 7-O-glucuronide, respectively. Using similar methods, compounds F3, F9 and F12 were tentatively identified as quercetin 7- O-glucoside, diosmetin 7O-rutinoside and kaempferol 3-O-(6”-malonylglucoside), respectively. [44–46]. 3.1.2. Phenylethanoid glycosides Seventeen phenylethanoid glycosides were identified in the BOEs. P1 showed an [M + Cl]− ion at m/z 335.0905 in negative ionization mode and an [M + Na]+ ion at m/z 323.1099 in positive ionization mode. The molecular formula C14 H20 O7 was calculated from the high resolution TOF-MS. The fragment ion at m/z 137 in negative ionization mode was attributed to loss of a glucose moiety from the [M + Cl]− ion, which further loses a molecule of H2 O to give a fragment at m/z 119. P1 was thus tentatively identified as salidroside by comparison with literature data [39]. P2 and P3showed similar MS spectra. Their deprotonated molecular ions [M−H]− at m/z 487 in negative ionization mode suggested the same molecular formula, C21 H28 O13 , and indicated that they could be a pair of isomers. The successive elimination of rhamnopyranosyl and glucose moieties from the deprotonated molecular ions yielded fragments

at m/z 341 and 179, respectively. The fragments at m/z 179 and 161 supported the presence a caffeoyl residue. Since the E-form has been reported to elute earlier than the Z-form, P2 and P3 were tentatively identified as cistanoside F and 4-O-(3,4-dihydroxy- Zcinnamoyl)-3-O-l-rhamnopyranosyl-d-glucose, respectively [47]. P4 exhibited an [M−H]¯ion at m/z 505, suggesting the molecular formula C21 H30 O14 . The fragment at m/z 341, resulting from loss of 164 Da, suggested the existence of an open-loop glucose moiety. The fragments at m/z 179 and 161 were attributed to the loss of caffeoyl and hexose moieties, respectively. P4 was thus tentatively characterized as hebitol II [48]. P5 gave an [M + Na]+ ion at m/z 395, suggesting the molecular formula C17 H24 O9 . The fragments at m/z 364 [M + Na-31]+ and m/z 233 [M + Na-162]+ resulted from loss of a methoxyl group and a glucose moiety, respectively. P5 was tentatively identified as syringin by comparing the MS and UV spectra with literature data [49]. Using similar methods, compounds P6 and P7 were tentatively identified as ˇ-4-caffeoylglucose and 3-caffeoylquinic acid, respectively [50,51]. ¯ [M+H]+ ions at m/z 555 and 521, respecP8 showed [M + Cl]and tively, in negative and positive ionization modes, suggesting the molecular formula C22 H32 O14 . This compound had a similar fragmentation pattern to P4. In both cases, the loss of a glucose moiety yielded a fragment at m/z 355, which eliminated a hexose moiety and produced an ion at m/z 193. The daughter ion was identical to that of ferulic acid moiety. P8 was, therefore, tentatively iden¯ tified as globularitol [48]. P9 showed [M + Cl]and [M + Na]+ ions at m/z 481 and 469, respectively, in negative and positive ionization modes. The fragment at m/z 307 [M + Na-162]+ resulted from loss of a glucose moiety. Taking into account literature data and other information in the UV and MS2 spectra [52], P9 was tentatively identified as 2-phenylethyl 6-O-ˇ-d-glucopyranosyl-ˇd-glucopyranoside. P10 was identified definitively as echinacoside by comparison with authentic standards and by comparing the corresponding UV and MS spectra with literature data [53]. Using

290

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similar methods and by comparing their corresponding UV and MS spectra and elution times with literature data, P11, P12 and P13 were tentatively identified as isocampneoside II, campneoside II and phenethyl-ˇ-primeveroside [39,54,55]. P15, P17 and P18all showed a deprotonated molecular ion [M−H]− at m/z 623 and MS/MS fragments at m/z 461, 179 and 161, indicating a similar fragmentation pattern to that of P16 in negative ionization mode. Compound P16 were identified definitively as acteoside by comparison with the authentic standards and their corresponding UV and MS spectra. Taking into account both literature data and their retention times, P15, P17 and P18 were tentatively identified as forsythoside A, cis-acteoside and isoacteoside, respectively [39,56]. Using similar methods, compounds P14 and P19 were tentatively identified as hebeoside and globusintenoside (or a globusintenoside isomer), respectively [57,58].

moieties can be used to identify the type and position of the glycosyl moiety. C5 showed a precursor ion at m/z 675, which is 324 Da larger than the sodiated aglycone ion, indicating the presence of two glucosyl moieties or a gentiobiosyl moiety, since the product ion of gentiobiose at m/z 347 represented the base peak. By comparing the authentic standards and their corresponding UV and MS data with literature values [67], C5 was unambiguously identified as mono-gentiobiosyl-crocetin (crocin III). The precursor ions of the other two crocins, 648 Da (C3) and 486 Da (C4), were larger than the sodiated aglycone ion. For C4, the fragments ions at m/z 675 (Y0 +324) and 513 (Y0 +162) indicated a gentiobiosyl residue and a glucosyl residue, respectively, while C3 showed only the ion Y0 +324. C3 and C4 were, therefore, identified as bis-gentiobiosylcrocetin (crocin I) and gentiobiosyl-glucosyl-crocetin (crocin II), respectively [67].

3.1.3. Alkaloid compounds In the present work, seven alkaloids, all of which are coumaroylspermidine analogs, were detected in the BOEs. The MS/MS spectra of compounds A1–A2 and A6–A7 are shown in Fig. 3. To the best of our knowledge, this is the first time that this type of alkaloid has been discovered in B. officinalis or even in Buddleja plants. It has been reported that this type of alkaloid possesses inhibitory activity against HIV-1 protease or the serotonin transporter [59,60]. A6 gave a molecular ion [M+H]+ at m/z 584. Its major fragment ion at m/z 438 [M+H-146]+ was attributed to cleavage of the amide bond between the cinnamoyl and spermidine moieties. The fragment ions at m/z 292 and 147 could be explained by loss of the second and third cinnamoyl residues. The daughter ions at m/z 420 and 275 resulted from loss of NH4 and NH3 from the fragment ions at m/z 438 and 292, respectively. By comparing the authentic standards and their corresponding UV and MS spectra with literature data [61], A6 was identified definitively as N1 ,N5 ,N10 -(E)-tri-p-coumaroylspermidine. A3, A4 and A5 all showed a molecular ion [M+H]+ at m/z 584 and similar fragmentation patterns to that of A6, indicating that they were isomers of A6. Using a similar method to that used to identify A6, and by comparing the corresponding UV and MS spectra and elution times with literature data [61], A3, A4 and A5 were tentatively identified as N1 ,N5 ,N10 -(Z)-tri-p-coumaroylspermidine, N1 (Z)-N5 -(Z)-N10 -(E)-tri-p-coumaroylspermidine and N1 (E)-N5 (Z)-N10 -(E)-tri-p- coumaroylspermidine, respectively. A2 exhibited fragment ions at m/z 454 [M+H-146]+ , 308 [M+H-146-146]+ and 292 [M+H-146-162]+ , resulting from loss of cinnamoyl or caffeoyl moieties. The fragment ions at m/z 147 and 163 confirmed the presence of both cinnamoyl and caffeoyl residues. The MS spectrum and fragmentation behavior of A2 were identical to those reported in the literature for N1 -(E)-caffeoyl-N5 , N10 -di-p-(E)-coumaroyl spermidine [62]. A2 was, therefore, tentatively identified as N1 -(E)-caffeoyl-N5 ,N10 -di-p-(E)-coumaroyl spermidine [63]. Similarly, by comparing their chromatographic behaviors with literature data, A1 and A7 were tentatively identified as N1 ,N5 -di-[(E)-p-coumaroyl]-spermidine (or its isomer) and keayanidine A, respectively [38,64,65].

3.2. Method validation for HPLC fingerprint analysis

3.1.4. Other compounds One iridoid (C1), one organic acid (C2) and three carotenoids (C3–C5) were characterized in the BOEs. By comparing their UV and MS data with literature values, C1 and C2 were tentatively identified as 6-methoxyl catalpol and tuberonic acid glucose [47,66]. The three carotenoids (C3–C5) were identified as crocins, which had previously been reported to be the major pigments in B. officinalis flowers [5]. The MS/MS spectra of compounds C3–C5 are shown in Fig. 3. All three compounds exhibited an ion attributed to sodiated aglycon (Y0 ) at m/z 351, which corresponded to crocetin. The fragments arising from sequential loss of glucose or gentiobiose

Method validation for the HPLC fingerprint analysis was conducted as described in Section 2.4. The results are expressed as relative standard deviations (RSD) of the average relative retention times (RRT) and relative peak areas (RPA) of the 28 characteristic common peaks compared with the reference F16 (linarin). The variation of RRT and RPA in the precision, stability and reproducibility tests did not exceed 5% and we can conclude, therefore, that the HPLC fingerprint method is accurate and reliable. 3.3. HPLC fingerprints of B. officinalis Using the HPLC method that was established in our previous study, the peaks of the 11 chemical markers had satisfactory resolution (Fig. 4). The HPLC fingerprints of B. officinalis from 12 different producing areas were generated using professional software (Fig. 4A), and then the mean chromatographic fingerprint was obtained by analyzing the 12 samples (Fig. 4B). A total of 28 characteristic common peaks were found in the chromatogram, which were identified or tentatively identified as 15 flavonoids (F2, F4–F11, F14, F16, F18, F20, F22, 23), ten phenylethanoid glycosides (P2, P7, P8, P10–P12, P14, P16, P17, P19), one alkaloid (A6) and two other constituents (C3, C5). Linarin is one of the most important active constituents of B. officinalis and, since it is present at relatively high levels, F16 was chosen as the reference peak. The RRT and RPA of each characteristic peak were calculated with respect to the reference peak. The average values for the 12 B. officinalis samples are listed in Table S1. The RSDs of the RRT and RPA were in the range 0.01–0.17% and 15.43–35.10%, respectively, demonstrating that although the chemical constituents of B. officinalis collected from different areas were similar, there were distinct differences in content. When the similarity between the chromatographs of the 12 samples of B. officinalis and the mean chromatographic fingerprints were calculated, the similarity values were all >0.900 (Tables 1 and S2), also indicating that there is little difference in the chemical composition of B. officinalis grown in different producing areas. 3.4. Quantitative determination of 11 components in B. officinalis A method for the simultaneous determination of 11 components of B. officinalis has been established and was successfully employed to analyze 12 samples. From the results summarized in Table 3, it can be seen that linarin (flavonoid) and acteoside (phenylethanoid) are two major constituents of B. officinalis. The amount of linarin in all samples was greater than that stipulated in the 2015 edition of the Chinese Pharmacopeia (0.5%, 5 mg/g), and S2, S3, S6–S8 and S12 were also present in relatively large amounts (>16 mg/g). The amount of acteoside was in the range

G. Xie et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 283–295

291

Fig. 3. MS/MS spectra of the electrospray ionization-generated [M+H]+ precursor ions of the alkaloids (A1, A2, A6, A7) and [M + Na]+ precursor ions of crocins (C3–C5) in positive ion mode.

20–35 mg/g in all samples; samples collected from Tibet had the highest amount (42.36 mg/g) and samples collected from Anhui had the lowest amount (21.65 mg/g). Linarin has, to date, been the only marker compound stipulated in the Chinese Pharmacopoeia for the quality control of B. officinalis, despite the fact that the amount of acteoside is approximately 2-fold greater than that of linarin. Acteoside has been shown to have remarkable activity on the nervous system and immune system, especially in senile diseases (Alzheimer’s disease) and autoimmune disease (slow nephritis), where an obvious therapeutic effect has been observed [68–70]. It is thus critical to simultaneously analyze linarin and acteoside in order to guarantee the quality of B. officinalis flowers.

N1 ,N5 , N10 - (E)-tri-p-coumaroylspermidine (alkaloid) and crocin III (carotenoid) are two other typical components. The sample from Sichuan contained the highest level of N1 ,N5 ,N10 -(E)-tri-pcoumaroylspermidine (2.33 mg/g) and that from Inner Mongolia contained the largest amount of crocin III (1.95 mg/g). The total amount of the 11 constituents varies among the different producing areas (Fig. 5). The total amount in samples from Guangxi (S12), Guizhou (S2), Sichuan (S3) and Tibet (S4) is the largest (>65 mg/g) while the samples from Anhui (S1) and Inner Mongolia (S10) have the lowest levels. These differences may arise because of different ecological environments and processing methods.

292

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Fig. 4. HPLC chromatographic fingerprints of 12 B. officinalis samples (A) and mean chromatographic fingerprint (B).

Table 3 The content of 11 compounds in B. officinalis from different habitats (Mean ± SD, mg/g, n = 3). Samples

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12

Contents (mg/g) a P10

F4

P16

F5

F11

F14

F16

A6

C5

F22

F23

3.96 ± 0.11 5.92 ± 0.22 6.83 ± 0.15 6.26 ± 0.03 3.81 ± 0.06 4.43 ± 0.16 3.86 ± 0.00 3.64 ± 0.03 8.37 ± 0.08 4.11 ± 0.08 4.28 ± 0.05 9.54 ± 0.19

1.18 ± 0.03 1.16 ± 0.02 1.34 ± 0.02 1.23 ± 0.02 0.89 ± 0.02 1.05 ± 0.02 1.09 ± 0.02 0.91 ± 0.02 0.69 ± 0.02 0.50 ± 0.01 0.95 ± 0.03 0.82 ± 0.01

21.65 ± 0.45 34.28 ± 0.44 29.75 ± 0.09 42.36 ± 0.11 26.79 ± 0.08 27.27 ± 0.43 25.89 ± 0.31 27.46 ± 0.29 29.59 ± 0.29 22.30 ± 0.23 26.86 ± 0.22 34.59 ± 0.48

0.73 ± 0.02 0.72 ± 0.01 0.54 ± 0.01 0.75 ± 0.01 0.62 ± 0.02 0.61 ± 0.03 0.79 ± 0.02 0.88 ± 0.02 0.85 ± 0.03 0.73 ± 0.01 0.60 ± 0.01 0.86 ± 0.04

1.07 ± 0.01 1.13 ± 0.05 1.24 ± 0.01 1.16 ± 0.01 1.08 ± 0.03 1.18 ± 0.01 1.10 ± 0.03 1.02 ± 0.02 0.95 ± 0.04 1.50 ± 0.02 1.13 ± 0.05 1.17 ± 0.01

2.20 ± 0.10 4.07 ± 0.13 3.27 ± 0.03 2.09 ± 0.05 2.33 ± 0.03 2.82 ± 0.12 2.47 ± 0.08 2.44 ± 0.05 1.50 ± 0.02 1.27 ± 0.02 2.64 ± 0.05 1.68 ± 0.04

13.68 ± 0.35 17.23 ± 0.49 17.69 ± 0.10 12.17 ± 0.09 14.03 ± 0.15 19.11 ± 0.35 16.18 ± 0.23 18.17 ± 0.19 13.23 ± 0.49 12.39 ± 0.14 15.26 ± 0.48 16.04 ± 0.15

1.29 ± 0.06 2.21 ± 0.09 2.33 ± 0.03 1.28 ± 0.00 1.37 ± 0.01 1.89 ± 0.07 1.05 ± 0.03 1.49 ± 0.06 1.55 ± 0.03 1.35 ± 0.02 1.58 ± 0.02 1.88 ± 0.02

0.39 ± 0.01 0.69 ± 0.02 1.54 ± 0.02 1.13 ± 0.01 1.32 ± 0.01 1.15 ± 0.02 1.74 ± 0.02 1.68 ± 0.01 0.48 ± 0.01 1.95 ± 0.02 1.10 ± 0.01 0.50 ± 0.00

0.56 ± 0.00 0.44 ± 0.01 0.81 ± 0.00 0.46 ± 0.01 0.59 ± 0.01 0.56 ± 0.02 0.69 ± 0.01 0.62 ± 0.02 0.37 ± 0.01 0.70 ± 0.00 0.49 ± 0.01 0.33 ± 0.00

0.21 ± 0.00 0.21 ± 0.00 0.24 ± 0.00 0.11 ± 0.00 0.25 ± 0.00 0.26 ± 0.00 0.17 ± 0.00 0.22 ± 0.00 0.15 ± 0.02 0.20 ± 0.00 0.21 ± 0.01 0.14 ± 0.00

3.5. Total phenolic content and evaluation of antioxidant activities TPC is widely used to assess antioxidant potential. TPCs of samples from different production areas are shown in Table 4. In the present study, the highest TPC was found in samples from Guizhou Province (39.83 ± 0.35 mg GAE/g), followed by samples from Guangxi Province. The sample from Shaanxi Province had the lowest TPC (33.26 ± 0.29 mg GAE/g). The TPCs of our samples were higher than those of a methanolic extract (28.82 ± 0.06 mg GAE/g)

described in another report [20]. These differences can likely be attributed to different extraction conditions. The general trend is for antioxidant activity to parallel TPC (Table 4). Correlation analysis showed a significant correlation between the DPPH, SOSA, FRAP and Fe2+ -chelating assays and TPC. The FRA showed the highest correlation (R2 = 0.863) and the SOSA showed the poorest correlation (R2 = 0.607). These results are in agreement with those described in an earlier report, in which plant material with a higher TPC showed stronger antioxidant activities. Taking into account all of the antioxidant test results, the

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293

Fig. 5. The total content of 11 compounds in B. officinalis from different habitats.

Table 4 Antioxidant activities of twelve batches of B. officinalis. NO

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12

Antioxidant activity (IC50) TPC (mg/g)

DPPH IC50 (mg/mL)

SOSA IC50 (ug/mL)

FRA EC50 (mg/mL)

Fe2+ -chelating IC50 (mg/mL)

33.67 ± 0.82 39.83 ± 0.35 36.69 ± 0.53 35.98 ± 0.48 33.52 ± 0.76 33.26 ± 0.29 35.95 ± 1.00 34.98 ± 0.46 37.71 ± 0.81 33.48 ± 0.49 37.69 ± 0.36 38.60 ± 0.44

0.64 ± 0.01 0.41 ± 0.01 0.44 ± 0.00 0.49 ± 0.01 0.59 ± 0.00 0.47 ± 0.01 0.46 ± 0.01 0.43 ± 0.01 0.52 ± 0.01 0.58 ± 0.01 0.48 ± 0.00 0.44 ± 0.01

258.82 ± 1.12 199.33 ± 2.34 184.85 ± 3.01 266.19 ± 3.84 212.72 ± 2.41 304.48 ± 1.29 278.35 ± 2.98 215.19 ± 3.70 232.71 ± 0.34 302.74 ± 5.63 227.74 ± 5.19 208.11 ± 4.31

1.124 ± 0.016 0.726 ± 0.016 0.810 ± 0.005 0.770 ± 0.001 0.935 ± 0.010 1.001 ± 0.005 0.871 ± 0.006 0.887 ± 0.001 0.809 ± 0.003 0.972 ± 0.012 0.834 ± 0.008 0.733 ± 0.004

2.40 ± 0.02 1.93 ± 0.01 1.97 ± 0.03 2.59 ± 0.01 2.39 ± 0.01 3.04 ± 0.02 2.60 ± 0.04 2.54 ± 0.02 2.61 ± 0.03 2.58 ± 0.01 2.08 ± 0.01 1.84 ± 0.01

methanol extract of B. officinalis was found to have good antioxidant activity and to be a potential natural antioxidant. The samples from Guizhou, Sichuan and Guangxi Province showed the greatest antioxidant capacity.

4. Conclusion This study provides chemical profiles of methanol extract of B. officinalis flowers and characterizes a total of 54 chemical compounds, including 23 flavonoids, 19 phenylethanoid glycosides, 7 alkaloids and 5 other constituents of the herb. Among them, 35 compounds, comprising 14 flavonoids, 11 phenylethanoid glycosides, 7 alkaloids, 1 organic acid and 2 carotenoids, were found firstly in B. officinalis. An accurate and reliable HPLC fingerprint method was developed to evaluate the quality of B. officinalis. For the fingerprint analysis, 28 characteristic fingerprint peaks were used to assess the similarities among 12 samples collected from different areas. These showed good similarity between the samples. The amount of 11 constituents in the 12 samples was also determined. Samples from Guangxi, Guizhou, Sichuan and Tibet were found to have relatively high amounts compared with samples from other areas. This is the first study to combine HPLC fingerprint analysis with the simultaneous determination of 11 bioactive compounds for quality evaluation of B. officinalis. The TPC and antioxidant capacities of B. officinalis flowers from 12 different habitats were measured using DPPH, SOSA, FRA and Fe2+ -chelating assays. B. officinalis was found to have good antioxidant capacity and to be a potential natural antioxidant. The highest antioxidant capacities were found in samples from

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